Vascular lesions and pneumonia in a pig fetus infected by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) associated reproductive disease was diagnosed in a herd containing only gilts. A single case of abortion occurred and no other disorder was evident in the herd. PCV2 antigen and/or DNA were detected in two aborted fetuses. One of the fetuses, revealing both PCV2 DNA and antigen, presented multinucleated giant cells, severe vascular lesions (intramural oedema, fibrinoid necrosis, mild lympho-histiocytic vasculitis, fibrin thrombi) and mild non-suppurative inflammation in the lungs. Other abortifacient infections were not found. This is the only report of PCV2-induced abortion in Hungary since 1999, when PCV2-associated disease was first discovered in the country.

Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism.

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log(10) titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.

Genome sequence of a monoreassortant H1N1 swine influenza virus isolated from a pig in Hungary.

The genome of a porcine H1N1 influenza A strain is reported in this study. The strain proved to be a monoreassortant strain with a typical porcine N1 gene on the genetic backbone of the pandemic H1N1 influenza A virus strain. Monitoring of descendants of the pandemic 2009 H1N1 strain is needed because of concerns that more-virulent strains may emerge in forthcoming epidemic seasons.

Typhlocolitis associated with spirochaetes in duck flocks.

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.

Porcine circovirus type 3 detection in a Hungarian pig farm experiencing reproductive failures.

Porcine circovirus 3 (PCV3) infection has been reported in piglets and sows with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. Few studies linked PCV3 infection to increased incidence of abortion and weak-born piglets. This is the first report of a detection of PCV3 Hungarian strain in several organs of aborted and weak-born piglets, including the thymus, lymph node, placenta, spleen, kidney and the liver. The tissue tropism of PCV3 in affected litters was analysed using real-time quantitative PCR, and the result showed the highest load of viral DNA in the thymus and lymph nodes. The ORF2 of Hungarian PCV3 strains was 524 nucleotides in length, and the sequence identity to GenBank sequences ranged from 98.5 per cent to 99.2 per cent. The results suggest that PCV3 may have a relevant role in reproductive failure in gilts.

Characterisation of the first complete genome sequence of the roe deer (Capreolus capreolus) papillomavirus.

The complete genomic DNA of a novel roe deer (Capreolus capreolus) papillomavirus (CcPV1) was amplified and sequenced from fibropapillomatous skin lesions of a Hungarian roe deer. Viral DNA was detected by a pair of degenerate primers and the remaining genomic sequence was amplified by a long-template high-fidelity PCR and sequenced. The CcPV1 genome was 8032 bp long and contained open reading frames (ORFs) typical for Delta-papillomaviruses (E6, E7, E1, E2, E4, E5, E9, L2, and L1) and a 799 bp long untranslated regulatory region (URR). Phylogenetic analysis based on the 3861 bp long concatenated sequence of the E1-E2-L2-L1 ORFs and on separate alignments of all major ORFs using both neighbour-joining and maximum parsimony methods placed CcPV1 on a distinct branch between Ovine papillomavirus 1 and the other deer papillomaviruses within the Delta-papillomavirus genus, although pairwise nucleotide alignments of L1 ORF sequences determined highest identities with European Elk Papillomavirus (71.2%) and Reindeer Papillomavirus (70.3%).

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Vector-borne transmission of Besnoitia besnoiti by blood-sucking and secretophagous flies: epidemiological and clinicopathological implications.

BACKGROUND: Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease. FINDINGS: Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents. CONCLUSIONS: Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

Detection of Brucella canis-induced reproductive diseases in a kennel.

Brucella spp. were isolated from an abortion case submitted for laboratory examination 8 months after the first clinical symptoms appeared in a kennel consisting of 31 dogs. Pathological investigations revealed the parallel presence of necrotic placentitis and the strong immunostaining of trophoblast cells by immunohistochemistry (IHC) using hyperimmune rabbit anti-Brucella canis primary antibodies. The rapid slide agglutination test was positive in 7 of 31 (23%) cases. The organism B. canis was successfully cultured from the blood, tissues, or vaginal swabs of only 3 of 31 (10%) cases. The isolated strains were identified as B. canis based on their colony morphology and agglutination with R sera. The strains were initially misidentified as B. suis with the "Bruce-ladder" method, and were subsequently correctly identified as B. canis with a single nucleotide polymorphism (SNP) typing test. Three culture-positive cases and 3 culture-negative cases with histories of reproductive disorders were selected and examined for the presence of B. canis infection using histopathology, IHC, and polymerase chain reaction (PCR) assays. Characteristic histologic lesions were found in all of the 6 animals, whereas IHC and PCR yielded positive results only in single cases from both groups. The results imply that all cases of canine abortion should be examined for brucellosis by bacterial culture of aborted fetuses and placentas. Immunohistochemical examination of placentas is also recommended because it is a quick and sensitive technique compared with bacterial culture. Multiple methods (i.e., serology, blood, and genital bacterial cultures) should be applied simultaneously and repeatedly for the reliable screening of B. canis infection in live individuals.

Endemic papillomavirus infection of roe deer (Capreolus capreolus).

Roe deer papillomavirus (CcPV1) infection has been identified as an endemic disease in roe deer populations of the Carpathian basin in Central Europe (Hungary, Austria and Croatia). The disease is characterised by easily recognizable skin tumours similar to deer papillomavirus infection of North American deer species. In 2006, a questionnaire study was conducted among all Hungarian game management units (GMUs) in order to assess the distribution of the disease and its major epidemiological features. Categorical information was collected about disease occurrence, trend and frequency of detection, on primarily affected age classes in both sexes, and association of lesions with mortality. Replies were received from 539 GMUs representing 50.9% of total GMU territory and disease presence was reported by 295 (54.7%) GMUs. Older age classes of both sexes were found to be more affected. Association of various environmental factors with disease occurrence was evaluated and data were collected on the occurrence of similar skin lesions in other European countries. Pathological features of CcPV1 infection were described and the localisation of both CcPV1 antigen and DNA was characterised by immunohistochemistry and in situ DNA hybridisation in skin lesions. Virus presence was also demonstrated by PCR and PCR product sequencing.

A 45-nucleotide insertion in the NS2 gene is responsible for the cytopathogenicity of a bovine viral diarrhoea virus strain.

Cytopathogenicity (cp) markers have recently been investigated in the genomes of field isolates of bovine viral diarrhoea virus (BVDV). Most of the isolates originated from mucosal disease (MD) cases observed after vaccination with a live attenuated vaccine, termed here BVDV-X. The NS2-3 genes of these isolates and of the vaccine proved to be identical, including a 45-nucleotide (nt) viral insertion at nt position 4355. The insertion originated from the NS4B/5A junction region of the BVDV genome. Interestingly, in BVDV strain CP7 a 27-nt insertion originating from the NS2 is located exactly at the same position. Complete genome analysis of BVDV-X did not reveal further potential cp markers. Furthermore, expression studies indicated that the insertion promotes NS2-3 cleavage. In order to examine the possible role of the 45-nt insertion in viral cytopathogenicity in details, a full-length infectious cDNA clone of BVDV-X was generated, and bovine turbinate (BT) cells were transfected with RNA transcribed from the clone. The recovered virus, termed BVDV-XR, showed slight retardation in growth in comparison with the original BVDV-X, and induced cytopathogenic effect (CPE). Since the natural non-cytopathogenic (ncp) counterpart of the vaccine virus was not available, an insertion-negative mutant cDNA clone was generated from BVDV-XR by PCR-directed mutagenesis. The recovered virus, termed BVDV-XR-INS-, showed the same growth characteristics as its cp counterpart BVDV-XR, but caused no CPE. These findings provide a direct proof that the 45-nt insertion at position 4355 has a basic role in the cytopathogenic character of this BVDV strain.

Diagnostic application of immunoperoxidase monolayer assay using monoclonal antibodies produced against equine arteritis virus 14-kDa nucleocapsid protein.

Two monoclonal antibodies against the Bucyrus strain of equine arteritis virus (EAV) were produced, and according to immunoperoxidase reaction following Western blot of electrophoresed EAV structural proteins, they recognized the nucleocapsid (N) protein antigen (14-kDa protein). Besides reacting with the blotted polypeptide, the antibodies of the two clones (designated 1H1 and 4G6) selected from 576 have shown high affinity and specificity to intracellular virus antigen as well. Both antibodies reacted with the representatives of the different subtypes of equine arteritis virus providing a suitable general tool for diagnostic purposes using immunoperoxidase monolayer assay (IPMA). Isotypes of the antibodies were examined by Ouchterlony immundiffusion assay. The subtyping of the two examined MAbs proved that the light chains are of the kappaisotype, whereas the heavy chains were identified as IgG 1 isotype.